The nervous system, like the immune system, develops from multipotent progenitor cells. The existence of neural progenitor cells that generate multiple types of neurons and glia has been well documented both in vivo and in vitro in the CNS and PNS (for reviews, see McKay, R. D. G., Cell 58:815-821 (1989); Sanes, J. R., Trends Neurosci. 12:21-28 (1989); McConnell, S. K., Ann. Rev. Neurosci. 14:269-300 (1991)). In some cases, moreover, such multipotent cells have been shown to be capable of self-renewal at the single-cell level (Stemple, D. L. and Anderson, D. J., Cell 71:973-985 (1992); Wren, D., Wolswijk, G. and Noble, M., Journal of Cell Biology. 116:167-176 (1992); Davis, A. and Temple, S., Nature 372:263-266 (1994) suggesting that they may be analogous to self-renewing hematopoietic stem cells (Spangrude, G. J., Heimfeld, S. and Weissman, I. L., Science 241:58-62 (1988)). In support of this idea, there is evidence in the CNS for the persistence of some kinds of neuronal and glial progenitors into adulthood (Altman, J., J. Comp. Neurol. 137:433-458. (1969); Kaplan, M. S. and Hinds, J. W., Science 197:1092-1094 (1977); Wolswijk, G. and Noble, M., Development 105:387-400 (1992); Reynolds, B. A. and Weiss, S., Science 255:1707-1710 (1992); Lois, C. and Alvarez-Buylla, A., Proc. Natl. Acad. Sci. USA 90:2074-2077 (1993); Morshead, C. M., Reynolds, B. A., Craig, C. G., McBurney, M. W., Staines, W. A., Morassutti, D., Weiss, S. and van der Kooy, D., Neuron 13:1071-1082 (1994)).
The existence of multipotent neural progenitors raises the question of how these cells generate their differentiated derivatives. On the one hand, cell fate could be assigned by lineage or by other cell-autonomous mechanisms. On the other hand, cell fate could be influenced or determined by cell-extrinsic signals. A popular idea to explain hematopoiesis is that both types of mechanisms operate, so that multipotent stem cells generate progenitors committed to one or more sublineages, which then proliferate, survive and differentiate in response to specific growth factors (Ogawa, M., Blood 81:2844-2853 (1993)). Similar “neuropoietic” models have also been invoked to explain cell lineage diversification in the nervous system (Anderson, D. J., Neuron 3:1-12 (1989); Sieber-Blum, M., In: Comments Developmental Neurobiology 1:225-249 (1990); LeDouarin, N., Dulac, C., Dupin, E. and Cameron-Curry, P., Glia. 4:175-184 (1991), although evidence in support of such models has been relatively scant and indirect (for review, see Anderson, D. J., Curr. Opin. Neurobiology 3:8-13 1993).
The neural crest represents a good model system in which to investigate the process of neural cell lineage diversification in vertebrates because it is relatively simple and experimentally accessible (LeDouarin, N. M., Cambridge University Press. Cambridge, UK (1982)). In vivo lineage-tracing studies (Bronner-Fraser, M. and Fraser, S., Nature 335:161-164 (1988); Frank, E. and Sanes, J. R., Development 111 pp 895-908 (1991) and in vitro clonal analyses (Sieber-Blum, M. and Cohen, A., Devel. Biol. 80:96-106 (1980); Baroffio, A., Dupin, E and Le Douarin, N. M., Proc. Natl. Acad. Sci. USA 85:5325-5329 (1988); Stemple, D. L. and Anderson, D. J., Cell 71:973-985 (1992); Ito et al., 1993) have demonstrated that many neural crest cells are multipotent at the time they emigrate from the neural tube in both avian and mammalian embryos. In the rate, moreover, serial cell cloning experiments have shown that such multipotent cells are capable of at least limited self-renewal in vitro (Stemple, D. L. and Anderson, D. J., Cell 71:973-985 (1992). Furthermore, the fate of such multipotent cells can be influenced by environmental signals (for review, see Stemple, D. L. and Anderson, D. J., Devel. Biol. 159:12-23 (1993)).
These experiments did not address the issue of whether neural crest cells undergo progressive restrictions in developmental potential. That such restrictions may occur has been suggested from studies of transplanted or cultured avian neural crest cell populations (Le Lievre, C. S., Schweizer, G. G., Ziller, C. M. and Le Douarin, N. M., Developmental Biology 77:362-378 (1980); Le Douarin, N. M., Science 231:1515-1522 (1986); Artinger, K. B. and Bronner-Fraser, M., Dev. Biol. 149:149-157 (1992) or from clonal analysis of postmigratory crest cells in peripheral ganglia (Duff, R. S., Langtimm, C. J., Richardson, M. K. and Sieber-Blum, M., Dev. Biol. 147:451-459 (1991); Hall, A. K. and Landis, S. C., Neuron 6:741-752 (1991); Deville, F. S.-S. C., Ziller, C. and Le Douarin, N., Dev. Brain Res. 66:1-10 (1992); Deville, F. S.-S. C., Ziller, C. and Le Douarin, N. M., Dev. Biol. 163:141-151 (1994). However, in the transplantation studies that manipulated the cells' environment, there was no analysis of single cells, and in the single cell culture experiments, there was no manipulation of the cells' environment. To date, there has been no study in which postmigratory neural crest cells in clonal culture have been challenged by exposure to environmental signals known to influence the fate of early migratory cells.
c-RET is an orphan receptor tyrosine kinase is one of the earliest surface markers that distinguishes postmigratory from early migrating neural crest cells (Pachnis, V., Mankoo, B. and Costantini, F., Development 119, in press.; Lo, L., Guillemot, F., Joyner, A. L. and Anderson, D. J., Persp. Dev. Neuro. 2:191-201 (1994)). RET is not simply a marker for enteric progenitors but is also essential for their proper development, as shown by genetic studies in both mice (Schuchardt, A., D'Agati, V., Larsson-Blomberg, L., Costantini, F. and Pachnis, V., Nature 367:380-383 (1994)) and humans (Edery, P., Lyonnet, S., Mulligan, L. M., Pelet, A., Dow, E., Abel, L., Holder, S., Nihoul-Fekete, C., Ponder, B. A. J. and Munnich, A., Nature 367:378-380 (1994). In situ hybridization experiments have indicated that RET is not expressed by early migrating trunk neural crest cells in vivo but is expressed after these cells have aggregated to form the primordia of autonomic ganglia (Pachnis, V., Mankoo, B. and Costantini, F., Development 119, in press.).
Both Ret and Mash1 are regulatory genes essential for the development of subsets of autonomic neurons, as shown by targeted gene disruption experiments in mice (Guillemot, F. and Joyner, A. L., Mech. Devel. 42:171-185 1993); Schuchardt, A., D'Agati, V., Larsson-Blomberg, L., Costantini, F. and Pachnis, V., Nature 367:380-383 (1994)). In addition, both genes are initially expressed in otherwise morphologically and antigenically undifferentiated neural crest cells (Lo, L., Johnson, J. E., Wuenschell, C. W., Saito, T. and Anderson, D. J., Genes & Dev. 5:1524-1537 (1991).; Guillemot, F. and Joyner, A. L., Mech. Devel. 42:171-185 (1993); Pachnis, V., Mankoo, B. and Costantini, F., Development 119, in press.). While Ret is genetically essential for the development of all enteric neurons, the precise developmental operation it controls is not yet established.
The fact that Ret and Mash1 are expressed sequentially (Guillemot, F. and Joyner, A. L., Mech. Devel. 42:171-185 (1993); Lo, L., Guillemot, F., Joyner, A. L. and Anderson, D. J., Persp. Dev. Neuro. 2:191-201 (1994)) in the same cells and that both are required for the differentiation of at least a subpopulation of peripheral autonomic neurons raises the possibility that there is an interaction between these two genes. For example, signally through RET could lead to the expression of MASH1; conversely, MASH1 could be required for the maintenance or up-regulation of RET expression. However, though Ret is required for the differentiation of all enteric neurons (Schuchardt, A., D'Agati, V., Larsson-Blomberg, L., Costantini, F. and Pachnis, V., Nature 367:380-383 (1994)), it is not essential for the initial differentiation of sympathetic neurons. Conversely, Mash1 is required for sympathetic neuron differentiation (Guillemot, F. and Joyner, A. L., Mech. Devel. 42:171-185 (1993)) but not for the differentiation of some enteric neurons. These data suggest that Mash1 expression does not require Ret function in sympathetic neurons, and that Ret function does not require Mash1 expression in late-generated enteric neurons. Nevertheless, recent evidence indicates that early-generated enteric neurons, including the serotonergic subset, require Mash1 function (Blaugrund et al., submitted) as well as Ret function (Schuchardt, A., D'Agati, V., Larsson-Blomberg, L., Costantini, F. and Pachnis, V., Nature 367:380-383 (1994)). This leaves open the possibility that there is a genetic interaction between Ret and Mash1 within this enteric sublineage. The ability to isolate RET+ neural crest cells from embryos of various genotypes would permit a more detailed analysis of the functions and interactions of these and other regulatory genes involved in neural crest development, as well as of the mechanistic basis of developmental restriction within this population.
Accordingly, it is an object of the invention to provide methods and compositions for the enrichment and characterization of neural progenitor cells.